Correlating structural modifications of small molecules with changes in their protein binding affinities is a fundamental problem in chemical biology. The difficulty associated with predicting energetics in protein-ligand interactions is exacerbated by an acute lack of detailed experimental data pertaining to how specific variations in ligand structure affect compensating changes in binding enthalpies and entropies in protein-ligand interactions. Contributing to the complexity of the problem is a paucity of information regarding how variations in ligand structure affect protein dynamics in protein-ligand complexes and whether differential changes in such dynamics have a significant effect upon binding energetics. Toward developing a better understanding of molecular recognition in biological systems, we have adopted a unique, multidisciplinary approach in which synthetic organic chemistry, microcalorimetry, protein crystallography, NMR spectroscopy, and computational chemistry are integrated in systematic studies to investigate explicitly how specific variations in ligand structure affect energetics, structure and dynamics in protein-ligand interactions in a well-defined biological system. Briefly, we will design and synthesize pseudopeptides that are derived from pTyr-Val-Asn and vary in their rigidity and/or preorganization, hydrophobicity, cation-@ stabilizing ability, and hydrogen bond accepting ability. The thermodynamic parameters for binding of these pseudopeptides to the Grb2 SH2 domain will be determined using isothermal titration calorimetry, and free energies of solvent transfer of representative compounds will be determined by solvent partition experiments. The consequences of varying ligand structure upon structure and dynamics of the protein in the complex will be studied by X-ray crystallography and NMR. Molecular dynamics simulations will be conducted using experimental data to refine the models and methods, so we can calculate relative binding energetics and probe changes in protein dynamics that occur upon binding ligands having different structures and affinities. The results will be analyzed, and correlations between specific changes in ligand structure with variations in thermodynamic binding parameters and protein flexibility will be identified so it can be ascertained whether changes in ligand structure can be correlated with changes in binding enthalpies and entropies and whether changes in flexibility of the Grb2 SH2 domain contribute significantly to the energetics of ligand binding. Insights obtained from these studies will be used to design second generation pseudopeptides having higher binding affinities for the Grb2 SH2 domain as these will be useful as tools for signal biology and as potential therapeutic leads. PUBLIC HEALTH RELEVANCE: The highly integrated experiments proposed herein are uniquely designed to determine how specific changes in ligand structure affect enthalpies, entropies and dynamics in protein-ligand complexes. The results of these studies will enhance our knowledge of molecular recognition in biological systems and contribute to developing experimental and computational methods that will facilitate the structure-based design of small molecules having high affinities for protein targets. Such tools are indispensable to medicinal chemists as they optimize ligand binding affinities and transform novel leads into selective and potent therapeutic agents to treat diseases.